20 November 2009. The DTNBP1 gene encoding dystrobrevin binding protein 1, otherwise known as dysbindin, has come up positive in schizophrenia patients in numerous genetic association studies around the globe, as well as in meta-analyses (see SZGene DTNBP1). Different genetic variants in and around the gene associate with the positive and negative symptoms of the disease, and scientists hope that by studying the protein they can learn something about the underlying pathology. But to date, very little is known about the protein or how mutations in the gene might impact the brain. Two papers published this week may help. In today’s Science, researchers report that dysbindin helps regulate the release of the neurotransmitter glutamate from fruit fly neurons. A paper in this week’s PNAS suggests that the protein modulates recycling of dopamine receptors on the cell surface of mouse cortical neurons and that it enhances the excitability of interneurons that release γ-aminobutyric acid (GABA). Since perturbations in glutamatergic, dopaminergic, and GABAergic neurons have all been implicated in the pathology of schizophrenia, together the papers raise the tantalizing thought that dysbindin is a player at the nexus of pathology.

At first blush, however, the two papers seem to contradict each other. The role described in flies is a presynaptic one, whereas that in the Sandy mouse, which has a deletion of a section of the DTNB1 gene, is a postsynaptic one. This adds to a growing debate about where dysbindin exerts most influence. In reality, the protein might work both pre- and postsynaptically especially since there are three distinct isoforms in humans that could perform very different roles.

The Sandy mouse model and postsynaptic dysbindin
Sandy mice have a naturally occurring mutation in the gene encoding dysbindin, which is absent in animals homozygous for the mutation. As reported in the PNAS paper, researchers led by Bai Lu at the National Institutes of Health, Bethesda, Maryland, looked at the role of dopamine neurotransmission in the Sandy mouse, which exhibits rodent equivalents of some of the behavioral symptoms of schizophrenia (e.g., psychomotor agitation and cognitive deficits). First author Yuanyuan Ji and colleagues found elevated numbers of dopamine D2 receptors on cortical neurons cultured from these animals, suggesting that dysbindin normally keeps those receptors from accumulating. Faulty dopaminergic neurotransmission is thought to be a key facet of schizophrenia, and dopamine D2 receptor antagonists are currently the most widely used drugs for treating psychoses.

What makes the D2 receptors accumulate? The first clue is that the total level of D2 receptors was not altered, suggesting normal synthesis and degradation of D2 protein. Ji and colleagues thought that uptake and recycling of the receptors might be compromised, but in experiments where they labeled and tracked cell surface proteins, they found the rate of D2 uptake in wild-type and dysbindin-negative mice was the same. However, they found that those receptors taken up from the cell surface were recycled in the cytoplasm and reinserted back into the cell membrane at up to threefold higher rates in dysbindin-negative neurons. The results suggest that dysbindin normally tempers the recycling of D2 receptors.

What effect would enhanced recycling of D2 receptors have on neurotransmission in the brain? The researchers recorded neurological activity in the mouse equivalent of the human dorsolateral prefrontal cortex (DLPFC), an area of the brain consistently linked to neurotransmission defects in schizophrenia. Ji and colleagues looked at several cell types in PFC slices, including pyramidal cells and fast-spiking interneurons. (There is evidence that both cell types may malfunction in schizophrenia.) Though they found that the Sandy mouse pyramidal cells seemed normal, the fast-spiking dysbindin-negative neurons were not so fast after all, producing significantly fewer spikes than their wild-type counterparts. The same was true for fast-spiking interneurons in the striatum. Whether or not this was due to altered recycling of D2 receptors was unclear. In addition, the researchers found that interneuron-driven GABAergic inhibitory currents in pyramidal cells were reduced in PFC slices from Sandy mice compared to wild-type slices. Together, the results suggest that reduced activity of GABAergic interneurons in Sandy mice leads to reduced inhibition of PFC pyramidal cells, which is a scenario that many researchers believe is playing out in the brains of people with schizophrenia.

Konrad Talbot, University of Pennsylvania, Philadelphia, who was not involved in the study, found the work very interesting. “This is probably the first study in print that demonstrates that fast-spiking interneurons in the Sandy mouse are quite abnormal,” he told SRF, and he noted how the findings may explain cognitive deficits in this mouse. “These neurons help drive γ oscillations in the brain, which are critical for cognitive function. If you disrupt them, you disrupt cognition,” he said. He also noted that cognitive defects in schizophrenia are possibly the most debilitating and are closely connected to the negative symptoms, for which there is currently no effective treatment.

Whether the changes seen in the Sandy mouse relate mechanistically to what is going on in schizophrenia remains to be seen, but Talbot’s group recently showed that levels of dysbindin protein are reduced in the DLPFC in schizophrenia cases. Interestingly, they found reductions in only one isoform of the protein, dysbindin-1C (see Tang et al., 2009). That isoform is located almost exclusively with postsynaptic proteins.

What to make of presynaptic dysbindin in the fruit fly?
There is evidence, however, that dysbindin plays a presynaptic role as well. David Jentsch and colleagues at the University of California, Los Angeles, reported that DLPFC pyramidal cells in the Sandy mouse are poor at a phenomenon called paired-pulse facilitation and have reduced postsynaptic currents, both indicative of dysfunctional presynaptic release of glutamate. Reported in today’s Science, the findings from Graeme Davis’s lab at the University of California, San Francisco, support that idea.

Davis and first author Dion Dickman carried out a genetic screen to look for proteins that help stabilize neurotransmission in the fruit fly (Drosophila) neuromuscular junction (NMJ). In the Drosophila NMJ, which uses glutamate as a neurotransmitter, blocking postsynaptic receptors with philanthotoxin-433 (PhTx) evokes a compensatory release of neurotransmitter from nerve terminals in a process dubbed homeostasis. One of the mutations that compromised homeostasis was an insertion in the fly homolog of dysbindin.

Talbot noted that there is only 28 percent sequence identity between human and Drosophila dysbindin, but he thinks Dickman and Davis’s work provides evidence that the fruit fly protein might be similar to human dysbindin-1B, which is found in presynaptic terminals. “This report introduces Drosophila as a model system for exploring what may be a basic function of presynaptic dysbindin-1, one which needs to be tested with mammalian homologs,” he told SRF.

The researchers found that in the fly dysbindin mutants there is a complete loss of NMJ homeostasis without any effect on baseline neurotransmission. Furthermore, when they expressed normal dysbindin in neurons, it corrected for the loss, indicating that the problem lay on the presynaptic side of the NMJ. They found no evidence for any morphological abnormality at the NMJs, which had the correct levels, location, and organization of synaptic markers.

By tagging dysbindin with a yellow fluorescent protein, the researchers found that the protein localizes with synaptic vesicle proteins. What dysbindin does at synaptic vesicles is unclear, but the researchers did find that the protein may work downstream of calcium signaling, which is required for synaptic neurotransmitter release in the fly NMJ. Evidence for that came from reducing availability of extracellular calcium and using mutants of the voltage-gated calcium channel “cacophony,” which is required for synaptic vesicle release at the NMJ. Reducing extracellular calcium significantly impaired baseline neurotransmission in dysbindin mutant neurons compared to wild-type, while in the same cells, overexpression of cacophony could not rescue loss of homeostasis. In contrast, enhanced synaptic release caused by overexpression of normal dysbindin still occurred in cacophony mutant flies. The results suggest that dysbindin may act downstream (or perhaps independently) of calcium signaling in presynaptic terminals.

Talbot warned that it cannot be assumed that dysbindin-1 in fruit flies has the same protein-protein interactions as dysbindin-1 in humans (or even mice). “Hence, you cannot assume that a function of dysbindin-1 found in Drosophila will be found in humans,” he said. He suggested the value of this work is the identification of a dysbindin-1 function that would be difficult to establish in other experimental systems. “Further testing will be needed to see if it is shared by presynaptic dysbindin-1 in mammals,” he said.—Tom Fagan.

Over the past few years, specific disruptions in the function of presynaptic, glutamate-releasing terminals in the cortex of animals with genetic insufficiency in dysbindin have been hypothesized and found in mammalian preparations (Talbot et al., 2004; Numakawa et al., 2004; Chen et al., 2008; Jentsch et al., 2009). Setting out to discover genes involved in presynaptic function in Drosophila, Dickman and Davis provide powerful convergent evidence supporting this biological role for the dysbindin protein. The seemingly similar functions for this protein in mammalian cortical synapses and at the invertebrate neuromuscular junction is an exciting finding, though one that should not be interpreted without caution.

Overall, the presynaptic defects that result from loss of dysbindin expression could be the basis of failures of sustained network activity in cortical regions that subserve representational knowledge and working memory-like processes. On the other hand, increasing attention is being focused on the consequences of dysbindin loss for components of the post-synaptic zone. Impaired receptor trafficking and alterations in cell excitability have been reported in pyramidal cells and fast-spiking cells (Ji et al., 2009; Jentsch et al., 2009).

Much remains unknown. What are the molecular mechanisms by which alterations in receptor trafficking are altered in post-synaptic targets? Are these cell autonomous effects or changes secondary to particular disturbances in network function caused by presynaptic dysfunction? Are pyramidal cells and/or particular subsets of interneurons more impacted?

Moreover, if there are disturbances in expression of particular isoforms of dysbindin, are these effects due to genetic variation within the DTNBP1 locus, or are these genomic phenotypes a result of transcriptional or translational influences on DTNBP1 expression?

It is clear that the biology of this gene and its associated protein is of great interest. Increasingly sophisticated tools that allow cell-type-specific regulation and/or modulation of expression in an isoform specific manner are required to help elucidate the answers to these questions.